Constitutive high expression of chromosomal beta-lactamase in Pseudomonas aeruginosa caused by a new insertion sequence (IS1669) located in ampD

Abstract

The expression of chromosomal AmpC beta-lactamase in Pseudomonas aeruginosa is negatively regulated by the activity of an amidase, AmpD. In the present study we examined resistant clinical P. aeruginosa strains and several resistant variants isolated from in vivo and in vitro biofilms for mutations in ampD to find evidence for the genetic changes leading to high-level expression of chromosomal beta-lactamase. A new insertion sequence, IS1669, was found located in the ampD genes of two clinical P. aeruginosa isolates and several biofilm-isolated variants. The presence of IS1669 in ampD resulted in the expression of high levels of AmpC beta-lactamase. Complementation of these isolates with ampD from the reference P. aeruginosa strain PAO1 caused a dramatic decrease in the expression of AmpC beta-lactamase and a parallel decrease of the MIC of ceftazidime to a level comparable to that of PAO1. One highly resistant, constitutive beta-lactamase-producing variant contained no mutations in ampD, but a point mutation was observed in ampR, resulting in an Asp-135-->Asn change. An identical mutation of AmpR in Enterobacter cloacae has been reported to cause a 450-fold higher AmpC expression. However, in many of the isolates expressing high levels of chromosomal beta-lactamase, no changes were found in either ampD, ampR, or in the promoter region of ampD, ampR, or ampC. Our results suggest that multiple pathways may exist leading to increased antimicrobial resistance due to chromosomal beta-lactamase.

FIG. 1.

Total sequence of the new insertion sequence, IS1669. The shaded 17-bp sequences in the upstream and downstream parts of IS1669 represent the “left inverted repeated sequence” and the “right inverted repeated sequence,” respectively. The inverted repeated sequences were imperfect in one nucleotide position (underlined). The amino acid composition of a probably 476-amino-acid transposase is shown.

FIG. 2.

Results of E-test measurements for the MIC of ceftazidime for three P. aeruginosa isolates with or without ampD complementation. The isolate 17107B-2 was AmpD defective since IS1669 was found in ampD. This was not the case for strains 19676A-2 or PAO1. ampD from PAO1 was used for complementation on the plasmid pNB101. Control (no complementation) was performed by transforming the isolates with pNB100 containing no ampD.